CONSTRUCTION OF A PHYSICAL MAP OF HUMAN CHROMOSOME 21

人类 21 号染色体物理图谱的构建

• 批准号: 3333868
• 负责人: DAVID PATTERSON
• 金额: $ 38.6万
• 依托单位: ELEANOR ROOSEVELT INST FOR CANCER RES
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3333868
• 关键词: artificial chromosomes; chromosomes; endonuclease; genetic library; genetic mapping; human genetic material tag; human tissue; in situ hybridization; molecular cloning; nucleic acid probes; polymerase chain reaction; restriction mapping

The overall goal of this project is to generate a complete physical map of at least the long arm of human chromosome 21. This chromosome is important for several human genetic diseases, including Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, progressive myoclonus epilepsy, and probably leukemia. A set of yeast artificial chromosomes (YACs) covering about 20% of the long arm of chromosome 21 (21q) already has been generated in this laboratory on behalf of the chromosome 21 Joint YAC Screening Effort. Moreover, this laboratory has constructed a pulsed field gel map of 2lq spanning over 44 million base pairs of DNA, or over 90% of the estimated size of this chromosome arm. Reagents available to construct the map, in the form of a set of ordered, overlapping YACs spanning the chromosome include 3 different YAC libraries representing roughly 13-fold coverage of the human genome, 107 additional regionally mapped chromosome 21-specific DNA probes not yet used to screen YAC libraries, a very detailed Chinese hamster/human cell hybrid regional mapping panel for chromosome 21 generated and characterized in this laboratory and 2 microclone libraries from microdissected chromosome 21s comprising at least 740,000 chromosome 21 clones. Methods in place to be used include both PCR and hybridization based YAC library screening protocols, YAC end cloning by Vectorette PCR and alu-vector PCR, long-range restriction mapping of YACs, and fluorescence in situ hybridization (FISH) to metaphase chromosomes using YACs as probes to assess YAC chimerism. A new technique, RecA Assisted Restriction Endonuclease (RARE) Cleavage, will be assessed to aid in validating the fidelity of the YAC contig, filling in gaps (closure), and for other mapping purposes.

这个项目的总体目标是生成一个完整的物理地图 至少是人类21号染色体的长臂。 这条染色体是 对包括唐氏综合症在内的几种人类遗传疾病很重要， 阿尔茨海默病、肌萎缩侧索硬化、进行性肌阵挛 癫痫，可能还有白血病 一组酵母人工染色体（YAC）覆盖约20%的 21号染色体长臂（21 q）已经在这一过程中产生。 实验室代表染色体21联合YAC筛选工作。 此外，本实验室还构建了2lq的脉冲场凝胶图， 跨越超过4400万个DNA碱基对，或超过90%的估计 可用于构建图谱的试剂， 跨越染色体的一组有序重叠的YAC的形式 包括3个不同YAC文库，代表大约13倍的覆盖率 在人类基因组中，107个额外的区域定位染色体 21-特异性DNA探针尚未用于筛选YAC文库， 详细的中国仓鼠/人细胞杂交区域图谱 染色体21产生和特点，在这个实验室和2 来自显微切割的21号染色体的微克隆文库， 至少740，000个染色体21克隆。 可用的方法包括 基于PCR和杂交的YAC文库筛选方案， 通过Vectorette PCR和alu载体PCR克隆，长距离限制性内切酶切 YAC的映射，和荧光原位杂交（FISH）， 使用YAC作为探针来评估YAC嵌合性。 一 新技术，RecA辅助限制性内切酶（RARE）切割， 将被评估以帮助验证YAC重叠群的保真度， 填补空白（关闭），并用于其他绘图目的。